Beta-arrestin 2 is required for lysophosphatidic acid-induced NF-kappaB activation

Abstract

Lysophosphatidic acid (LPA) is a bioactive phospholipid and binds to its receptors, a family of G protein-coupled receptors (GPCR), which initiates multiple signaling cascades and leads to activation of several transcription factors, including NF-kappaB. Although LPA-induced signaling pathways have been intensively investigated, the molecular mechanism by which LPA activates NF-kappaB is not fully defined. In this work, we found that beta-arrestin 2, but not beta-arrestin 1, is required for LPA-induced NF-kappaB activation and interlukin-6 expression. Mechanistically, we found that beta-arrestin 2 associated with CARMA3, a scaffold protein that plays an essential role in GPCR-induced NF-kappaB activation, suggesting that beta-arrestin 2 may recruit CARMA3 to LPA receptors. Although beta-arrestin 2 deficiency did not affect LPA-induced IKKalpha/beta phosphorylation, it impaired LPA-induced IKK kinase activity, which is consistent with our previous findings that CARMA3 is required for IKKalpha/beta activation but not for the inducible phosphorylation of IKKalpha/beta. Together, our results provide the genetic evidence that beta-arrestin 2 serves as a positive regulator in NF-kappaB signaling pathway by connecting CARMA3 to GPCRs.

Fig. 1.

β-Arrestin 2 is required in LPA- and PMA/Iono-induced NF-κB activation and in cytokine production. (A) Wild-type and βarr1/2 dKO MEF cells were stimulated with or without LPA (10 μM) or PMA (40 ng/ml) plus Ionomycin (100 ng/ml) for 60 min or TNFα (10 ng/ml) for 30 min. Nuclear extracts were prepared and subjected to EMSA by using ³²P-labeled NF-κB or OCT-1 probes. (B) Wild-type, βarr1 KO, or βarr2 KO MEF cells were stimulated as in A. Nuclear extracts were prepared and subjected to EMSA by using ³²P-labeled NF-κB or OCT-1 probes. (C) Wild-type or βarr2 KO MEF cells were serum-starved for 4 h and stimulated with or without LPA (10 μM), PMA (40 ng/ml) plus Iono (100 ng/ml) (P/I), or TNFα (10 ng/ml) for the indicated time points. Supernatants were collected, and IL-6 concentrations in the supernatants were measured by ELISA. All data were normalized to the value of unstimulated control in respective cells as the fold induction. Error bars indicated ±SD between triplicate experiments.

Fig. 2.

LPA- and PMA/Iono-induced IκBα phosphorylation and IKK kinase activity are dependent on β-arrestin 2. (A–C) Wild-type, βarr1 KO, and βarr2 KO MEF cells were stimulated with LPA (10 μM) (A), PMA (40 ng/ml) plus Ionomycin (100 ng/ml) (B), or TNFα (10 ng/ml) (C) for indicated time points. Phosphorylation of IκBα and/or IKK was examined by Western blotting with the indicated antibodies. (D) Wild-type or βarr2 KO MEF cells were stimulated with or without LPA (10 μM), PMA (40 ng/ml) plus Ionomycin (100 ng/ml) (P/I), or TNFα (10 ng/ml) for the indicated time points. The IKK complex was immunoprecipitated by using a mixture of IKKα and IKKγ antibodies and protein A–agarose. The immunoprecipitated complex was subjected to an in vitro kinase assay with GST-IκBα (1–62) as substrates. Parts of the lysates of the immunoprecipitated IKK complex and GST-IκBα (1–62) substrates were subjected to Western blotting as loading controls.

Fig. 3.

β-Arrestin 2, but not β-arrestin 1, can rescue the defect of LPA-induced NF-κB activation in βarr1/2 dKO cells. βArr1/2 dKO MEF cells were reconstituted with a retroviral vector encoding FLAG-tagged β-arrestin 1, β-arrestin 2, or empty vector, respectively. The resulting cells were stimulated with or without LPA, PMA/Iono, or TNFα. The nuclear extracts from these cells were subjected to EMSA with ³²P-labeled NF-κB and OCT-1 probes. Cytosolic extracts were subjected to SDS/PAGE and Western blotting with the indicated antibodies.

Fig. 4.

Interaction of CARMA3 with β-arrestins. (A) HEK293T cells were transfected with expression vectors encoding HA-tagged CARMA3, FLAG-tagged β-arrestin 1, or FLAG-tagged β-arrestin 2 at different combinations. Twenty hours after transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads, and immunoprecipitated proteins and cell lysates were subjected to SDS/PAGE and analyzed by immunoblotting with the indicated antibodies. (B) Wild-type or β-arrestin 2 KO MEF cells (15-cm plate, 80% confluent) were stimulated with or without LPA for different periods of time and then scraped off and lysed. The resulting lysates were subjected to immunoprecipitation with CARMA3 antibody-conjugated beads. The obtained immunocomplexes were subjected to SDS/PAGE and Western blotting with β-arrestin 1 and β-arrestin 2 antibodies or CARMA3 antibodies as indicated. For detection of coimmunoprecipitated β-arrestins, rabbit IgG TrueBlot was used as secondary antibody.

Fig. 5.

The domain of CARMA3 interacted with β-arrestin 2. (A) Schematic diagram of full-length and deletion mutants of CARMA3. CC, coil-coiled domain; SH3, Src homology 3 domain; PDZ, PDZ domain; GUK, guanylate kinase domain. (B) FLAG-tagged full-length or deletion mutants of CARMA3 together with HA-tagged β-arrestin 2 was transfected into HEK293T cells. Twenty hours after transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads, and immunoprecipitated proteins and cell lysates were subjected to SDS/PAGE and analyzed by immunoblotting with the indicated antibodies.

Fig. 6.

β-Arrestin 2 links CARMA3 to LPA receptor. (A) Expression vectors encoding HA-β-arrestin 2, HA-LPA₁, and FLAG-CARMA3 at different combinations were transfected into HEK293T cells. Twenty hours after transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads, and immunoprecipitated proteins and cell lysates were subjected to SDS/PAGE and analyzed by immunoblotting with the indicated antibodies. (B) Working model for β-arrestin 2-mediated NF-κB activation. GPCR (LPA receptor)-induced NF-κB activation involves in the β-arrestin-dependent recruitment of CARMA3 to the receptor. The recruitment of CARMA3 leads to the formation of a complex containing Bcl10, MALT1, and TRAF6. This complex may regulate polyubiquitination or unknown modification of the IKK complex, whereas a β-arrestin 2- and CARMA3-independent, but PKC-dependent signal induces IKK phosphorylation by an unknown kinase in the GPCR signaling pathway. In the absence of β-arrestin 2, CARMA3 is not recruited into the LPA receptor complex. Therefore, CARMA3-dependent regulation of IKK complex is impaired, which results in the defect of IKK and NF-κB activation.